High-throughput screening (HTS) is one of the most powerful unbiased approaches to identify small molecule modulators of distinct biochemical activities. For this, assays are developed for an enzyme of interest and up to millions of compounds are screened to identify novel drug leads. Most HTS assays rely on labels?fluorescent or chemical?as a readout for activity. Labels are now known to introduce artifacts and false positives that slow down progress and drive up costs. Moreover, not all enzymes are amenable to label-based approaches, contributing to the lack of drugs for many clinically relevant targets. This proposal offers a universal solution by developing a novel label-free technology that could be applied to screen any biochemical activity using native substrates. The approach uses self-assembled monolayers (SAMs) of alkanethiolates on gold presenting a 3-trifluoromethyl-3-phenyl-diazirine (TPD) group that upon irradiation generates a reactive carbene that inserts into a broad range of chemical bonds to covalently immobilize the analyte of interest. A key advantage of using SAMs on gold is their compatibility with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) that provides a label-free and rapid, quantitative readout of immobilized analytes. The technique, termed traceless immobilization (TI)-SAMDI offers a novel, powerful tool that makes all enzymes ?screenable,? opening new therapeutic avenues for challenging and previously intractable targets.